The present invention relates generally to the repair of cartilage and the treatment of cartilagenous disorders.
Cartilagenous disorders broadly describe a collection of diseases characterized by degeneration of or metabolic abnormalities in the connective tissues which are manifested by pain, stiffness and limitation of motion of the affected body parts. The origin of these disorders can be pathological or as a result of trauma or injury.
Osteoarthritis (OA), also known as osteoarthrosis or degenerative joint disease, is the result of a series of localized degenerative processes that affect the articular structure and result in pain and diminished function. The incidence of OA increases with age, and evidence of OA can be detected at least one joint in the majority of the population by age 65. OA is often accompanied by a local inflammatory component that may accelerate joint destruction.
OA is characterized by disruption of the smooth articulating surface of cartilage, with early loss of proteoglycans (PG) and collagens, followed by formation of clefts and fibrillation, and ultimately by full-thickness loss of cartilage. Coincident with the cartilagenous changes are alterations in periarticular bone. The subchondral bone thickens and is slowly exposed. Bony nodules or osteophytes also often form at the periphery of the cartilage surface and occasionally grow over the adjacent eroded areas. OA symptoms include local pain at the affected joints, especially after use. With disease progression, symptoms may progress to a continuous aching sensation, local discomfort and cosmetic alterations such as deformity of the affected joint.
In contrast to the localized nature of OA, rheumatoid arthritis (RA) is a systemic, inflammatory disease which likely begins in the synovium, the tissues surrounding the joint space. The prevalence of RA is about ⅙ that of OA in the general population of the United States. RA is a chronic autoimmune disorder characterized by symmetrical synovitis of the joint and typically affects small and large diarthrodial joints, leading to their progressive destruction. As the disease progresses, the symptoms of RA may also include fever, weight loss, thinning of the skin, multiorgan involvement, scleritis, corneal ulcers, the formation of subcutaneous or subperiosteal nodules and premature death. While the cause of RA and OA are distinctly different, the cytokines and enzymes involved in cartilage destruction appear to be similar.
Because mature chondrocytes have little potential for replication, and since recruitment of other cell types is limited by the avascular nature of cartilage, mature cartilage has limited ability to repair itself. For this reason, transplantation of cartilage tissue or isolated chondrocytes into defective joints has been used therapeutically. However, tissue transplants from donors run the risk of graft rejection as well as possible transmission of infectious diseases. Although these risks can be minimized by using the patient""s own tissue or cells, this procedure requires further surgery, creation of a new lesion in the patient""s cartilage, and expensive culturing and growing of patient-specific cells. Better healing is achieved if the subchondral bone is penetrated, either by injury/disease or surgically, because the penetration into the vaculature allows recruitment and proliferation of undifferentiated cells to effect repair. Unfortunately, the biochemical and mechanical properties of this newly formed fibrocartilage differ from those of normal hyaline cartilage, resulting in inadequate or altered function. Fibrocartilage does not have the same durability and may not adhere correctly to the surrounding hyaline cartilage. For this reason, the newly synthesized fibrocartilage may be more prone to breakdown and loss than the original articular hyaline cartilage tissue.
Peptide growth factors are very significant regulators of cartilage growth and cartilage cell (chondrocyte) behavior (i.e., differentiation, migration, division, and matrix synthesis or breakdown) F. S. Chen et al., Am J. Orthop. 26: 396-406 (1997). Growth factors that have been previously proposed to stimulate cartilage repair include insulin-like growth factor (IGF-1), Osborn, J. Orthop. Res. 7: 35-42 (1989); Florini and Roberts, J. Gerontol. 35: 23-30 (1980); basic fibroblast growth factor (bFGF), Toolan et al., J. Biomec. Mat. Res. 41: 244-50 (1998); Sah et al., Arch. Biochem. Biophys. 308: 137-47 (1994); bone morphogenetic protein (BMP), Sato and Urist, Clin. Orthop. Relat. Res. 183: 180-87 (1984); Chin et al., Arthritis Rheum. 34: 314-24 (1991) and transforming growth factor beta (TGF-xcex2), Hill and Logan, Prog. Growth Fac. Res. 4: 45-68 (1992); Guerne et al., J. Cell Physiol. 158: 476-84 (1994); Van der Kraan et al., Ann. Rheum. Dis. 51: 643-47 (1992). Treatment with peptide growth factors alone, or as part of an engineered device for implantation, could in theory be used to promote in vivo repair of damaged cartilage or to promote expansion of cells ex vivo prior to transplantation. However, because of their relatively small size, growth factors are rapidly absorbed and/or degraded, thus creating a great therapeutic challenge in trying to make them available to cells in vivo in sufficient quantity and for sufficient duration.
The present invention proposes to overcome this limitation by delivery of a growth factor with a vehicle, and/or as a slow-release formulation. The ideal delivery vehicle is biocompatible, resorbable, has the appropriate mechanical properties, and results in no harmful degradation products.
Another method of stimulating cartilage repair is to inhibit the activity of molecules which induce cartilage destruction and/or inhibit matrix synthesis. One such molecule is the cytokineIL-1xcex1, which has detrimental effects on several tissues within the joint, including the generation of synovial inflammation and up-regulation of matrix metalloproteinases and prostaglandin expression. V. Baragi, et al., J. Clin. Invest. 96: 2454-60 (1995); V. M. Baragi et al., Osteoarthritis Cartilage 5: 275-82 (1997); C. H. Evans et al., J. Leukoc. Biol. 64: 55-61 (1998); C. H Evans and P. D. Robbins, J. Rheumatol. 24: 2061-63 (1997); R. Kang et al., Biochem. Soc. Trans. 25: 533-37 (1997); R. Kang et al., Osteoarthritis Cartilage 5: 139-43 (1997). One means of antagonizing IL-1xcex1 is through treatment with soluble IL-1 receptor antagonist (IL-1ra), a naturally occurring protein that prevents IL-1 from binding to its receptor, thereby inhibiting both direct and indirect effects of IL-1 on cartilage. Other cytokines, such as IL-1xcex2, tumor necrosis factor alpha (TNF-xcex1), interferon gamma (IFN-xcex3), IL-6 and IL-8 have been linked to increased activation of synovial fibroblast-like cells, chondrocytes and/or macrophages. The inhibition of these cytokines may be of therapeutic benefit in preventing inflammation and cartilage destruction. In fact, molecules which inhibit TNF-xcex1 activity have been shown to have potent beneficial effects on the joints of patients with rheumatoid arthritis.
Nitric oxide also likely plays a substantial role in destruction of cartilage. [Ashok et al., Curr. Opin. Rheum. 10: 263-268 (1998)]. Unlike normal joint tissue which does not produce NO unless stimulated with cytokines such as IL-1, synovial membranes or cartilage obtained from arthritic joints spontaneously produce large amounts of nitric oxide for up to 3 days after removal from the joint. In addition, increased concentrations of nitrites are found in synovial fluid of arthritic patients. In addition to its direct stimulation of cartilage catabolism, nitric oxide present in an inflamed joint would likely lead to increased vasodilation and permeability, further release of cytokines such as TNF-xcex1 and IL-1 from leukocytes, and stimulation of angiogenesis. Evidence for a causative role of NO in arthritis, comes from animal models where inhibition of NO has been shown to prevent IL-1 mediated cartilage destruction and chondrocyte death as well as progression of osteoarthritis. Finally, several agents (such as auranofin, glucocorticoids, cyclosporins, tetracyclines, and at least some nonsteroidal anti-inflammatory drugs including aspirin) currently used for the treatment of human rheumatic diseases, have been shown to reduce NO production and/or activity.
Prior studies in diabetic (with altered serum insulin levels) or abnormal (hypophysectomized) animals suggests that insulin is required for optimal production of sulfated mucopolysaccharides and collagen, two major components of connective tissue. As early as 1957, insulin was shown to increase the otherwise abnormally low uptake of labeled sulfate into the skin of diabetic rats Schiller and Dorfman, J. Biol. Chem. 227: 625-632 (1957). Similarly, insulin increased the otherwise low level of sulfate uptake in aortae from diabetic rats. Cohen, M. P. and Foglia, V. G. Proc. Soc. Exp. Biol. Med. 132: 376-378 (1969); Proc. Soc. Exp. Biol. Med. 133: 1275-1278 (1970); Proc. Soc. Exp. Biol. Med. 135: 113-115 (1970). Subsequently, insulin was shown to stimulate uptake of sulfate into cartilage, but the identity of the molecules into which the sulfate was incorporated was not determined. Furthermore, the effect of insulin on endogenous matrix turnover (i.e. protein breakdown or retention within the matrix) was not assessed. Salmon, W. D., Jr., and Daughaday, W H. Endocrinol. 82: 493-499 (1957); J. Posever et al., J. Orthopaedic Res. 13: 832-827 (1995). While insulin may have direct effects on connective tissues, at least some data suggests that the defects in connective tissue metabolism found in diabetic animals, which can be at least partially reversed by systemic administration of insulin, could be due to circulating factor(s) induced by insulin and not due to direct effects of insulin on connective tissues (Spanheimer, R. G., Matrix 12: 101-107 (1992).
Insulin has also been found to stimulate the growth of mouse fibroblast cultures, Paul and Pearson, J. Endocrinol. 21: 287-294 (1960), cartilage cells from hypophysectomized rats (Salmon, W. D., Jr., J. Lab. Clin. Med. 56: 673-681 (1960), cells in bone cultures (Prasad, G. C. and Rajan, K. T., Acta Orthop. Scand. 41: 44-56 (1970), as well as cells in many other systems (Gey, G. O. and Thalhimer, W., J. Amer. Med. Assoc. 82: 1609 (1924); Lieberman, I., and Ove, P. O., J. Biol. Chem. 234: 2754-2758 (1959); Younger, L. R., King, J. and Steiner, O. F., Cancer Res. 26: 1408-1413 (1966); Schwartz, A. G. and Amos, H., Nature 219: 1366-1367 (1968). Most of these very early studies were performed on whole animals, organs, or tissues. Hajek and Solursh, Gene Comp. Endocrin. 25: 432-446 (1975) were among the first to show a direct effect of insulin on chondrocytes, derived from chick embryo sternal cartilage, in serum-free cultures. The stimulation of growth and mucopolysaccharide synthesis by insulin in these cultures may not be surprising given that the hormone insulin increases amino acid uptake, promotes a positive nitrogen balance, and favors overall protein synthesis. Similarly the increase in proteoglycan synthesis stimulated by insulin in rat tumor cells derived from a Swarm rat chondrosarcoma was accompanied by an increase in incorporation of radioisotope into total protein and thus may reflect a general increase in protein synthesis (Stevens, R. L. and Hascall, V. C., JBC 256:2053-2058 (1981). Finally, it should be understood that high levels of insulin (10 xcexcg/ml for the chick cultures, Hajek and Solursh, supra) were used in many of these studies. At such high concentrations insulin binds to, and activates, the insulin-like growth factor (IGF)-1 receptor, thus mimicking the effects of IGF-1 itself. Thus, the observed physiological effects could be the result of IGF-1 receptor signalling and merely the result of insulin signalling. Finally, systemic administration of insulin had no effect in an inflammatory, polyarthritic model in rats in which only the number of swollen joints was monitored. Roszkowski-Sliz, W., Acta Physiol. Pol. XXIV, 371-376 (1973). Since inflammation can occur in the absence of cartilage and bone destruction and vice versa in animal models, Joosten et al., J. Immunol. 163:5049-5055 (1999), how insulin may have affected the underlying joint tissues in this study is not clear.
More than thirty years ago, high doses of insulin were injected subcutaneously into 10 patients, 7 of whom had rheumatoid arthritis, with the goal of inducing a hypoglycemic crisis, increasing corticosteroid levels, and thus altering adrenal gland activity (M. Ippolito et al., Reumatismo 20(5): 561-64 (1967). Insulin treatment associated with cortisone resulted in overall beneficial effects for the patients including regain of appetite, an increase in body weight, and improvement in pain. These effects may be due to indirect activities of insulin, for example the ability of insulin to increase plasma corticosteroid levels Since the authors were most interested in the effects of systemic insulin on the xe2x80x9cdiencephalohypophysial systemxe2x80x9d, the anabolic effects of insulin on cartilage were not examined or considered. Furthermore, given the avascular nature of cartilage and the rapid clearance of insulin in vivo, it is unlikely that much if any of the subcutaneously-delivered insulin would have been available to chondrocytes within the joints of the insulin-treated individuals.
Unlike OA, bone loss is a common feature of RA. Japanese patent application JO 59-234,826, filed Nov. 7, 1984 also speculated on the potential application of insulin for the treatment of rheumatoid arthritis based on its induction of a bone marker, i.e. alkaline phosphatase activity, in the osteoblastic (bone) cell line, MC3T3-E1. However, the effects of insulin on cartilage tissue itself was not examined or considered.
As the population ages, and the incidence of arthritis increases, an effective therapy to induce repair of cartilage, including cartilage damaged as a result of injury and/or disease, is urgently needed.
The present invention concerns methods for the treatment, repair and protection of cartilage damaged as a result of a cartilagenous disorder, including that which results from disease and/or injury. More specifically, the invention concerns a method for the treatment, repair and protection of cartilage comprising administering an effective amount of insulin or an insulin variant. More specifically, the method provides for administration of insulin or insulin variant in a sustained- or extended-release form.
In a further embodiment, the present invention concerns a method for the treatment of cartilage damaged as a result of a cartilagenous disorder comprising contacting said cartilage with an effective amount of insulin and/or an insulin variant. Optionally, the cartilage is articular cartilage, and is contained within a mammal and the amount administered is a therapeutically effective amount. Optionally, the cartilagenous disorder is osteoarthritis, rheumatoid arthritis or an in injury.
In a further embodiment, the present invention concerns a method for the treatment of cartilage damaged by injury or preventing the initial or continued damage comprising contacting said cartilage with an effective amount of insulin or insulin variant. More specifically, the injury treated is microdamage or blunt trauma, a chondral fracture, an osteochondral fracture, or damage to tendons, menisci, or ligaments. More specifically, the cartilage is contained within a mammal, including humans, and the amount administered is a therapeutically effective amount. In a specific aspect, the injury can be the result of excessive mechanical stress or other biomechanical instability resulting from a sports injury or obesity. Alternatively, the present invention concerns a method of treating or facilitating the repair of bone fractures comprising contacting the region of the bone injury with an effective amount of insulin or insulin variant.
In a further embodiment, the present invention concerns a method for the treatment of cartilage damaged or preventing initial or continued damage by a cartilagenous disorder and/or injury comprising contacting said cartilage with an effective amount of a composition further comprising insulin or insulin variant. Alternatively, the composition further comprises a carrier, excipient or stabilizer. Alternatively, the cartilage is present in a mammal and the amount administered is a therapeutically effective amount. Alternatively, the composition may be administered via injection or infusion by intravenous, intraarterial, intraperitoneal, intramuscular, intralesional, intraarticular or topical administration to a mammal and the amount administered is a therapeutically effective amount. Alternatively, the composition is injected directly into the afflicted cartilagenous region or joint.
In a further embodiment, the present invention concerns a method for the treatment of cartilage damaged or preventing initial or continued damage by a cartilagenous disorder and/or injury comprising administrating a therapeutically effective amount of a sustained or extended-release composition containing insulin or insulin variant. Alternatively, the cartilage is present in a mammal and the amount administered is a therapeutically effective amount. More specifically, the extended- or sustained-release composition contains insulin or insulin variant formulated in a microencapsulation, a semi-permeable membrane of solid hydrophobic polymers, a biodegradable polymer(s), or a dispersion (e.g., suspension or emulsion). More specifically, the semi-permeable membrane of solid hydrophobic polymer is poly-lactic-co-glycolic acid (PLGA), and the biodegradable polymer is cross-linked hyaluronic acid (HA). Alternatively, the extended- or sustained-release insulin or insulin variant composition further comprises a water-soluble polyvalent metal salt. More specifically, the polyvalent metal salt includes the salt formed from a metallic cation and an inorganic or organic acid.
In a further embodiment, the invention concerns a method for treating cartilage damaged or preventing initial or continued damage as a result of injury or a cartilagenous disorders comprising contacting the cartilage with an effective amount of insulin or insulin variant in combination with an effective amount of a cartilage agent. Optionally, the cartilage is present inside a mammal and the amount administered is a therapeutically effective amount.
In another embodiment, the invention concerns a method of maintaining, enhancing or promoting the growth of chondrocytes in serum-free culture by contacting the chondrocytes with an effective amount of insulin or insulin variant. Alternatively, the method concerns contacting the chondrocyte with an effective amount of insulin or insulin variant in a sustained or extended-release formulation. Alternatively, the present invention concerns a method of stimulating the regeneration or preventing the degradation of cartilage resulting from injury or a cartilagenous disorder by transplantation of an effective amount of chondrocytes previously treated with an effective amount of insulin or insulin variant.
In a further embodiment, the present invention concerns a method for the treatment of cartilage damaged or preventing initial or continued damage as a result of injury or a cartilagenous disorder comprising contacting said cartilage with an effective amount of insulin and/or an insulin variant in combination with any standard cartilage surgical technique. The present invention may be administered prior, after and/or simultaneous to the standard cartilage surgical technique. In a specific aspect, the standard surgical technique may be selected from the following procedures: cartilage shaving, abrasion chondroplasty, laser repair, debridement, chondroplasty, microfracture with or without subchondral bone penetration, mosaicplasty, cartilage cell allografts, stem cell autografts, costal cartilage grafts, chemical stimulation, electrical stimulation, perichondral autografts, periosteal autografts, cartilage scaffolds, shell (osteoarticular) autografts or allografts, or osteotomy.
In a further embodiment, the invention concerns nucleic acid encoding insulin and/or insulin variants, and vectors and recombinant host cells comprising such nucleic acid.
In another embodiment, the present invention concerns a therapeutic kit, comprising insulin and/or an insulin variant and a carrier, excipient and/or stabilizer (e.g. a buffer) in suitable packaging. The kit preferably contains instructions for using insulin to treat cartilage damaged or to prevent initial or continued damage to cartilage as a result of a cartilagenous disorder. Alternatively, the kit may contain instructions for using insulin to treat a cartilagenous disorder.
In a further embodiment, the invention concerns an article of manufacture, comprising:
a container;
an instruction on the container; and
a composition comprising an active agent contained within the container;
wherein the composition is effective for treating a cartilagenous disorder, the instruction on the container indicates that the composition can be used to treat a cartilagenous disorder, and the active agent in the composition is an agent stimulating the repair and/or preventing the degradation of cartilage. In a preferred aspect, the active agent is insulin or an insulin variant.